Food and Chemical Toxicology 48 (2010) 3316–3320

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Food and Chemical Toxicology

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Study on antioxidant activity of common dry fruits

Neeraj Mishra ⇑, Akhilesh Dubey, Rahul Mishra, Nabneeta Barik
Department of Biotechnology, Saroj Institute of Technology and Management, Ahimamau, Sultanpur Road, Lucknow, Uttar Pradesh, India

a r t i c l e i n f o a b s t r a c t

Article history: This study investigates the antioxidant activity of different dry fruits (almonds, walnut, cashew nut,
Received 12 August 2009 raisins, chironji) through several chemical and biochemical assays: reducing power, lipid peroxidation
Accepted 20 August 2010 damage in biomembranes, determination of antioxidant enzymes activity (SOD and CAT). To estimate
the total phenolic content, the assay using Folin–Ciocalteu reagent was used. The EC50 values were
calculated for all the methods in order to evaluate the antioxidant efficiency of each dry fruit. The results
Keywords: obtained were quite heterogenous, revealing significant differences among the dry fruits. The methanolic
Dry fruits
extract of walnut showed the higher value of antioxidant activity based on lipid peroxidation assay. The
Lipid peroxidation
SOD
higher phenolic content was found in walnuts followed by almonds cashew nut, chironji and least
CAT phenolic content was found in raisins. Walnut revealed the best antioxidant properties, presenting lower
EC50 EC50 values in all assays except in antioxidant enzymatic activity.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction scavenging activity of antioxidants in foods has been substantially

Antioxidant compounds in food play an important role as a
health-protecting factor. Scientific evidence suggests that antioxi-
dants reduce the risk for chronic diseases including cancer and
heart diseases. Primary sources of naturally occurring antioxidants
are whole grains, fruits and vegetables.
Plant sourced food antioxidants like vitamin C, vitamin E, caro-
tenes, phenolic acids, phytate and phytoestrogens have been rec-
ognized as having the potential to reduce disease risk. Most of
the antioxidant compounds in a typical diet are derived from plant
sources and belong to various classes of compounds with a wide
variety of physical and chemical properties. Some compounds,
such as gallates, have strong antioxidant activity, while others,
such as the mono-phenols are weak antioxidants. The main charac-
teristic of an antioxidant is its ability to trap free radicals. Highly
reactive free radicals and oxygen species are present in biological
systems from a wide variety of sources. These free radicals may
oxidize nucleic acids, proteins, lipids or DNA and can initiate
degenerative disease. Antioxidant compounds like phenolic acids,
polyphenols and flavonoids scavenge free radicals such as perox-
ide, hydroperoxide or lipid peroxyl and thus inhibit the oxidative
mechanisms that lead to degenerative diseases. There are a num-
ber of clinical studies suggesting that the antioxidants in fruits,
vegetables, tea and red wine are the main factors for the observed
efficacy of these foods in reducing the incidence of chronic diseases
including heart disease and some cancers. The free-radical
⇑ Corresponding author. Tel.: +91 0522 281259; fax: +91 0522 2812760.
E-mail address: neerajhbti@gmail.com (N. Mishra).
investigated and reported in the literature by Miller et al.
(2000a,b). It has been established that oxidative stress is among
the major causative factors in induction of many chronic and
degenerative diseases including atherosclerosis, diabetes mellitus,
cancer, Parkinson’s disease and immune dysfunction and is
involved in aging (Halliwell, 2000; Metodiewa and Koska, 2000;
Young and Woodside, 2001). Antioxidants, both exogenous and
endogenous, can be effective in prevention of the free radical for-
mation by scavenging or promotion of their decomposition and
suppression of such disorders (Halliwell, 2000; Maxwell, 1995).
Free radicals and other reactive oxygen species such as super oxide
anion (O2 ), hydrogen peroxide and hydroxyl radical (OH ) in the
body are derived either from normal essential metabolic processes
or from external sources. Their chemical reactivity can damage all
types of cellular macromolecules including proteins, carbohy-
drates, lipids and nucleic acids. The importance of the antioxidant
constituents in the maintenance of health and protection from
coronary heart diseases and cancer is also raising interest among
scientists, food manufacturers and consumers as the trend of the
future is moving toward functional food with specific health effects
(Velioglu et al., 1998; Kahkonen et al., 1999; Robards et al., 1999).
Antioxidant activity (AOA) is a very important parameter used to
characterize different dry fruits materials. This activity is related
with compounds capable of protecting a biological system against
the potential harmful effect of oxidative processes. These reactions
cause excessive oxidation, involving reactive oxygen and nitrogen
species (RONS) (Halliwell, 1996; Krinsky, 1989). Excess free radi-
cals can result from a variety of condition such as tissue damage,
overexposure to environmental factors, a lack of antioxidants, or

0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.08.029
 N. Mishra et al. / Food and Chemical Toxicology 48 (2010) 3316–3320
destruction of free-radical scavengers (Victor et al., 2006). Then,
antioxidants are essential to preserve the biological system from
free radicals damage to biological molecules. Although the mam-
malian body has defence mechanisms to combat and to reduce
oxidative damage, epidemiological evidence indicate that the
consumption of food stuff containing antioxidant phytonutrients
and other poly phenolics is advantageous for health (Amarowicz
and Pegg, 2001). The additive and synergistic effects of such bioac-
tive molecules present in plant food are responsible for their
potent antioxidant properties (Pellegrini et al., 2006; Pereira
et al., 2006).
In the present study we report the total phenolic content and
antioxidant activity of different dry fruits such as almond (Prunus
amygdales), walnut (Juglans regia), cashew nut (Anacardium occi-
dentale L.), Raisins (Couma macrocarpa), chironji (Buchanania lan-
zan). Here, the antioxidant potential of various dry fruit extracts
were evaluated through several chemical and biochemical assays:
determination of antioxidant content, reducing power, lipid perox-
idation, determination of antioxidant enzymes through NBT test.
2. Materials and methods
2.1. Standard and reagents
Standards BHA (2-tert-butyl-4-methoxyphenol), L-ascorbic acid, a-tocopherol,
gallic acid, methanol, tris–HCl, ferric chloride, Folin–Ciocalteu reagent, sodium
dodecyl sulphate, thiobarbituric acid (TBA), sodium carbonate, ferrous sulphate,
n-butanol, potassium ferricyanide, sodium phosphate buffer, trichloroacetic acid,
ammonium sulphate, nitroblue tetrazolium, phenazine methosulphate, NADH,
hydrogen peroxide, distilled water.
2.2. Methanolic extraction
Dry fruits were brought from local market. For antioxidant compounds extrac-
tion, a fine powder (20 mesh) of samples (3 g each) was extracted using 50 ml of
methanol at 25 °C for 60 min. The extracts were filtered through Whatman No. 4
and evaporated at 40 °C to dryness. All the samples were redissolved in water at
concentration of 20 mg/ml.
2.3. Determination of phenolic content
Phenolic concentration in the extracts was estimated by colorimetric assay
based on procedures described by Singleton and Rossi (1965) with some modifica-
tions. Gallic acid was used for constructing the standard curve and the results were
expressed as mg of gallic acid equivalents/g extracts (GAEs).
2.4. Reducing potential
Concentration of dry fruits extracts (1 mg/ml, 2 mg/ml, 4 mg/ml, 6 mg/ml and
8 mg/ml) were mixed with 2.5 ml of 200 mmol/l sodium phosphate buffer (pH
6.5) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50 °C
for 20 min. After 2.5 ml of 10% trichloroacetic acid (w/v) were added, the mixture
was centrifuged at 1000 rpm for 8 min. The upper layer (5 ml) was mixed with
5 ml of distilled water and 1 ml of 0.1% of ferric chloride and the absorbance was
measured spectrophotometrically at 700 nm (Barros et al., 2007). The extract con-
centration providing 0.5 of absorbance was calculated from the graph of absorbance
at 700 nm against extract concentration. BHA and a-tocopherol were used as
standards.
2.5. Lipid peroxidation
Rats were anesthetized and subjected to intracardiac perfusion with ice-cold
saline in order to eliminate the excess of iron that could be released from intracel-
lular storage sites that otherwise may artificially increase free radical formation.
Brains were dissected on ice-chilled glass plates and the dissected sample was
immediately frozen on solid CO2. The samples were then homogenized in ice-cold
tris–HCl buffer (20 mM, pH 7.4) with a tissue homogenizer to produce a 1/10
homogenate. Two millilitres of brain homogenate (10%) was taken in a series of
35 mm petridishes to which extracts of various concentration (1 mg/ml, 2 mg/ml,
4 mg/ml, 6 mg/ml and 8 mg/ml) were added and mixed gently to form homoge-
neous solution. Lipid peroxidation was initiated by adding 100 ll of 15 mM ferrous
ammonium sulphate and thereafter petridishes were incubated at 37 °C. The FeSO4-
dependent system for 2-deoxyribose degradation results in formation of malondial-
dehyde (MDA) which is a TBA-reactive product. After 30 min, 100 ll of this reaction
mixture was taken in a tube containing 1.5 ml of 10% TCA. After 10 min tubes were
3317
centrifuged and supernatant was separated and mixed with 1.5 ml of 0.67% TBA in
50% acetic acid. The mixture was heated in a hot water bath at 85 °C for 30 min to
complete the reaction. The intensity of pink colored complex formed was measured
at 535 nm. The percentage inhibition of lipid peroxidation was calculated by com-
paring control (FeSO4 control) with treated (FeSO4 + extract) groups.
2.6. Superoxide dismutase
Two millilitres of brain homogenate was dispensed in centrifuge tube. The
tubes were placed in a refrigerated centrifuge and spinned at 10,000g for 15 min.
To the supernatant from each sample, 313 mg/ml ammonium sulphate was added
to the final concentration of 50%, the tubes were shaken thoroughly and kept for 4 h
in cold (4 °C). Thereafter, the tube was centrifuged at 14,000g for 30 min at 4 °C. The
supernatant sample was dialysed against cold tripled distilled water with three
changes, each change after 3 h interval. The content of dialysis bags were subse-
quently used as enzyme source. Later the two reaction setups run in parallel. The
tubes in first setup (experimental) received. 0.3 ml of 1.5 mM nitroblue tetrazolium
(NBT). 0.2 ml of 0.93 mM phenazine methosulphate, 1 ml of 20.4 mM pyrophos-
phate buffer pH 9.2, 1 ml distilled water and 0.2 ml enzyme source. The reaction
was started simultaneously in the two sets by the addition of 0.1 ml 2.34 mM
NADH. After an interval of 90 s 1 ml glacial acetic acid was added to each tube
for checking the reaction then 0.2 ml enzyme source was added in the reference
tubes the absorbance of these tubes was read at 560 nm on a spectrophotometer
against blank (NBT + PMS + buffer + TDW).
2.7. Catalase
Evaluation of catalase activity was carried out on the basis of conversion of
hydrogen peroxide into water and molecular oxygen described by Abei (1974).
2.8. Statistical analysis
For all the experiment three samples of each dry fruits were analyzed and all
the assays were carried out in triplet. The results are expressed as mean values
and standard error (SE) of the mean or standard deviation (SD) of the mean
(mean ± SD). The differences between the dry fruits were analyzed using one-way
analysis of variance (ANOVA) followed by t-test with p < 0.05.
3. Results and discussion
Percent yield of methanolic extracts of walnut (J. regia), almond
(P. amygdales), cashew nut (A. occidentale L.), chironji (B. lanzan)
and raisins (C. macrocarpa) were found to be 2.69, 2.52, 1.64,
1.23 and 0.46, respectively.
Total phenolic content of dry fruits have been presented in
Table 1. All dry fruits extracts showed significant value of total
phenols. It is comprehensible that walnut, almonds, cashew nut,
Chironji present the highest antioxidant activity. Walnut shows
highest phenolic content (31.66 ± 0.11 mg/g) whereas raisin shows
least but significant (p < 0.005) phenolic content among all the
extracts (Table 1). Walnut and almonds have been reported to be
rich in tannins (Amarowicz and Pegg, 2001); these phenolic
compounds might account for the values obtained as tannin
(phenolic component) is mainly gallic acid, consisting of 3,6-
digalloyglucose, pyrogallol, and resorcinol (Hwang et al., 2001).
Figs. 1–4 show the antioxidant activity of dry fruits extracts
examined as a function of their concentration. Several biochemical
assays were used to screen the antioxidant properties: reducing
Table 1
Phenolic contents of different extracts.
No. Scientific name English common Phenol contents
name (mg/g)
1. Juglans regia Walnut 31.66 ± 0.11
2. Prunus amygdales Almond 18.33 ± 0.11
3. Vitis vinifera Raisins 8.33 ± 0.11
4. Anacardium Cashew nut 16.33 ± 0.11
occidentale
5. Buchanania lanzan Buchanania 11.66 ± 0.57
Each experiment was repeated three times. Results have been expressed as
mean ± standard deviation.
3318 N. Mishra et al. / Food and Chemical Toxicology 48 (2010) 3316–3320
Fig. 1. Reducing power of different extracts and standards (BHA and a-tocopherol).
The absorbance at 700 nm was recorded in triplicate and each experiment was
repeated three times. The values are expressed as mean ± SD.
Fig. 2. Effect of varied concentrations of different extracts on Fenton reaction
mediated lipid peroxidation in mice brain homogenate. Each experiment was
performed in triplicate and was repeated three times.
Fig. 3. Effect of extracts on SOD activity. The inhibitory effect upon SOD is
measured as % inhibition in NBT reduction. The absorbance at 560 nm was recorded
in triplicate and experiments were repeated three times. Values are expressed as
mean ± SD.
power (measuring the conversion of a Fe3+/ferricyanide complex to
the ferrous form), inhibition of lipid peroxidation in brain tissue
(measured by the color intensity of MDA–TBA complex) and
Fig. 4. The effect of extracts on catalase activity. The decomposition of hydrogen
peroxide was observed directly by decrease in extinction at 240 nm, the difference
in extinction per unit time was taken for measurement of catalase activity.
measuring the antioxidant enzymatic activity (SOD and CAT). The
assays were performed for each extract separately. Nevertheless,
those assays were carried out using whole extracts instead of
individual compounds. According to Liu (2003), additive and
synergistic effects of phytochemicals in fruits and vegetables are
responsible for their potent bioactive properties and the benefit
of a diet rich in fruits and vegetables is attributed to the complex
mixture of phytochemicals present in whole foods. This explains
why no single antioxidant can replace the combination of natural
phytochemicals to achieve the health benefits. Analysis of Figs.
1–4 revealed that antioxidant activity increased with the concen-
tration, very good results being obtained, even at low extract
concentrations.
Some authors have reported a direct correlation between anti-
oxidant activity and total phenolic content (Velioglu et al., 1998;
Barros et al., 2007). The antioxidant activity of phenolic constitu-
ents may be related to their redox properties, which allow them
to act as reducing agents or hydrogen-atom donors, their ability
to chelate metals, inhibit lipoxygenase and scavenge free radicals
(Decker, 1997). Thus natural antioxidant function as free-radical
scavengers and chain breakers, complexers of pro-oxidant metals
ions and quenchers of single-oxygen formation (Amarowicz and
Pegg, 2001).
The reducing power also increased with concentration and the
values obtained for all the extracts were excellent (Fig. 1). The ef-
fect of methanolic extract on the reducing power was compared to
that of BHA and a-tocopherol in this study (Fig. 1). Dose response
studies indicated that before reaching a threshold level, there was
a positively linear relationship between the reducing power and
the concentration of different dry fruits. At 1 mg/ml, the absor-
bance values were above 0.65 for all extracts, once more with
the exception of walnut (1.26). The extracts obtained with cashew,
chironji showed similar values, while raisins showed less good re-
sults. It has been reported that the reducing properties are gener-
ally associated with the presence of reductones, which have been
Table 2
Maximum inhibition of Fenton reaction mediated lipid peroxidation in brain
homogenate by different dry fruits extracts.
No. Scientific Common name Max. Conc.
name inhibition (%) (mg/ml)
1. Juglans regia Walnut 66 8
2. Buchanania lanzan Buchanania 58 8
3. Vitis vinifera Raisins 49 8
4. Anacardium occidentale Cashew nut 61 8
5. Prunus amygdales Almond 58 8
 N. Mishra et al. / Food and Chemical Toxicology 48 (2010) 3316–3320 3319

Table 3
EC50 values (mg/ml) obtained in the antioxidant assays for dry fruits extract, and corresponding coefficient of variation (%).

Walnut Almonds Cashew nut Chironji Raisins

Reducing power assay
EC50 11.03 ± 0.36 12.08 ± 0..36 11.12 ± 0.38 19.16 ± 0..36 22.44 ± 0.32
CV (%) 22.32 27.37 33 34.59 30.76
Lipid peroxidation assay
EC50 2.25 ± 1.33 5.56 ± 1.45 8.20 ± 1.04 9.0 ± 0.86 11.45 ± 0.96
CV (%) 4.09 6.17 6 4.09 4.27
Superoxide dismutase (SOD)
EC50 14.57 ± 1.23 13.96 ± 1.15 14.53 ± 1.30 15.11 ± 1.53 18.40 ± 0.26
CV (%) 26.91 29 28.69 29.94 10.83
Catalase (CAT)
EC50 12.30 ± 0.60 11.50 ± 0.37 12.61 ± 0.24 13.10 ± 0.22 18.40 ± 0.26
CV (%) 26 24.66 9.1 10.47 10.83

Each experiment was repeated three times. Results have been expressed as mean ± standard deviation.

shown to exert antioxidant action by breaking the free radical
chain by donating a hydrogen atom (Shimada et al., 1992). Hence,
walnut and almonds may have high amounts of reductones. Anal-
ysis of variance showed that dry fruits extract has significant stron-
ger (p < 0.05) reducing power. This could be due to high amount of
total phenolic present in metabolic extract. It has to be reported
that walnut has showed highest reducing power and raisins has
least reducing power. Our result shows that walnut and almond
showed higher reducing power where raisins showed least reduc-
ing power.
Inhibition of lipid peroxidation was evaluated using thiobarbi-
turic acid-reactive substances (TBARS). This is a highly sensitive
method, the results being fully dependent on efficient centrifuga-
tion to remove the precipitated protein. Otherwise this will lead
to erroneous absorbance results. As can be easily understood from
Fig. 2, the capacity of inhibition of lipid peroxidation is propor-
tional to the extract concentration. This method permitted the
achievement of very high inhibition percentages at extremely
low concentrations. To verify this observation, we can note the
percentages obtained at 8 mg/ml: 68% (walnut), 61% (cashew),
58% (almonds), 58% (chironji) and 49% (raisins) (Table 2).
Table 3 shows antioxidant activity with EC50 values of walnut,
almonds, cashew nut, chironji, raisins measured by different
biochemical assays. Overall, walnut revealed the best antioxidant
properties (significantly lower EC50 values; p < 0.05). The EC50 val-
ues obtained for these extracts were excellent, particularly for LPO
inhibition. Raisins revealed a very poor antioxidant activity. Cash-
ew nut and almonds also revealed very good antioxidant activity,
while raisins presented the highest EC50 values in all the tested
method.
The EC50 values obtained for lipid peroxidation inhibition were
better than those for reducing power, SOD activity, CAT activity. All
the parameters assayed, present CV values that reveal high
reproducibility, ranging from 4.09% to 30.76%. In the case of reduc-
ing power, LPO inhibition, SOD activity and CAT activity, CV values
varied from 22.32% (walnut) to 34.59% (raisins), 4.09% (walnut) to
6% (cashew nut), 10.83% (raisins) to 29.94% (chironji) and 9.1%
(almonds) to 26% (walnut), respectively. Some other authors also
reported tree nuts have potential antioxidant activity, namely:
walnuts (Fukuda et al., 2003) and hazelnuts (Alasalvar et al.,
2006; Sivakumar and Bacchetta, 2005). No studies were reported
on chironji and raisins antioxidant properties.
As far as we know, this is the first report concerning the antiox-
idant activity of five different dry fruits extracts. The work herein
indicates that walnut and almonds present the highest antioxidant
activity values. The results obtained indicate a high potential of
application for these dry fruits extracts, traditionally considered
as important food products. They can be included in foods with
remarkable benefits for human or animal health.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors are grateful to Mr. Sunil Singh (Chairman),
Mr. Kaptan Singh (Advisor), Dr. Vinod Bihari (Director) & Mr. S.K.
Agarwal (Director Administration) from Saroj Institute of Technology
and Management, Lucknow for the encouragement and facilities
required for the experiments.
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